Liver Injury Associated with Metamizole Exposure: Features of an Underestimated Adverse Event

The data from 383 patients who were referred to the University Hospital Munich with acute liver injury and recruited for our ongoing prospective study on the effects of potentially hepatotoxic drugs (ClinicalTrials.gov: NCT 02353455) between March 2013 and July 2020 were analysed. Included in this study are healthy donors, patients prior to receiving therapy with drugs with DILI potential, patients with acute liver injury and DILI suspicion, as well as patients with acute liver injury as a result of other causes. Written informed consent was obtained from each patient. The study protocol conforms to the ethical guidelines of the Declaration of Helsinki and was approved by the Ethics Committee of the Faculty of Medicine, LMU Munich (project number 55-13). For the current analysis, 32 patients who had consumed metamizole were selected.

Acute liver injury was defined according to the consensus criteria from 2011: (a) ALT ≥ 5 × ULN, (b) alkaline phosphatase activity ≥ 2 × ULN, or (c) ALT ≥ 3 × ULN and TBIL ≥ 2 × ULN [12]. The ULN for aspartate aminotransferase (AST) activity and ALT were 35 U/L for women and 50 U/L for men, the ULN for alkaline phosphatase was 105 U/L for women and 130 U/L for men and the ULN for TBIL was 1.2 mg/dL. The type of liver injury was classified using the R ratio values, (ALT/ULN)/(alkaline phosphatase/ULN), with R ≥5 defining a hepatocellular injury, R ≤ 2 a cholestatic injury and 2 < R < 5 defining a mixed-type injury [13].

The diagnosis of DILI was based on clinical and laboratory findings, the RUCAM score [14], a causality assessment by the treating physician and expert opinion [15], as well as an in vitro test established in our centre using monocyte-derived hepatocyte-like cells (MH cells) [16‐18]. The interpretation of the RUCAM score is: < 0, drug is excluded as the cause; 1–2, unlikely; 3–5, possible; 6–8, probable; and > 8, highly probable. Regarding the RUCAM score for metamizole, 1 point was given for the item ‘known hepatoxicity’, as liver injury is not listed as an adverse event in the drug label of metamizole but has been described in the literature [4, 8]. A causality assessment by the treating physician was conducted and expert opinion from consulting hepatologists from our centre was used by consideration of all clinical data available, the course of events and by using the LiverTox database [19]. The assessment of causality was performed independently from the MH cell test result.

Blood samples for MH cell testing were acquired within 4 weeks after the onset of liver injury. Monocyte-derived hepatocyte-like cell generation and testing were performed as described previously [20]. Briefly, monocytes were isolated from patients’ blood samples and cultivated under serum-free conditions for 10 days, generating cells with some hepatocyte features such as cytochrome P450 activities. These cells, MH cells, are incubated for 48 hours in 96-well plates using 1 × maximum concentration (C_max) and 10 × C_max of the implicated drugs the respective patient had consumed. For the current analysis, MH cell testing was performed for every patient and metamizole was tested in 94% of the patients included along with the concomitant medications used by the respective patients. Metamizole was used in concentrations of 50 µM and 500 µM, which equals 1 × C_max and 10 × C_max of this drug. The lowest amount of cells used for a single test is 10,000 cells per well. All drugs are solved in a standardised medium (DMEM/HAM-F12 containing penicillin/streptomycin and 2 l-glutamine). Then, toxicity is measured with a standardised algorithm based on the release of lactate dehydrogenase in the supernatant and cell lysate. Results are normalised to a negative (0%) and positive control (100%), meaning lysis with 1% TWEEN^®20 (polyethylene glycol sorbitan monolaurate). To compensate for variance in seeding density, toxicity values are divided by two standard deviations (ULN) of the individual controls. The vector graphic demonstrating MH cell test results was created using Prism 8, Version 8.4.3.

The severity of the DILI episode was categorised as mild (1), moderate (2), severe (3) or fatal (4; death or the need for liver transplantation because of liver failure) according to the 2011 criteria of an international DILI expert group [12]. Acute liver failure was defined according to the American Association for the Study of Liver Diseases: (1) absence of pre-existing liver disease, (2) coagulopathy with an INR ≥ 1.5 in the absence of oral anticoagulants and (3) hepatic encephalopathy [21]. ‘Hy’s law’ criteria were fulfilled if ALT was ≥ 3 × ULN and TBIL was ≥ 2 × ULN, defining a subgroup of patients with DILI with a 10% risk of fatality or need of liver transplantation [22].

At enrolment, a thorough medical history was taken, including previous diseases, symptoms associated with liver injury, co-morbidities as well as current or previous medications. Each patient’s age, sex, ethnicity, height, weight and relevant data from clinical investigations were recorded. A thorough hepatological work-up, including liver function tests, serological studies for hepatitis A, B, C, D, and E, cytomegalovirus, Epstein–Barr virus and herpes simplex virus as well as autoantibody screening was performed, and results were extracted from the medical records. The presence of anti-nuclear antibodies (ANA) and other liver-specific antibodies was tested using an indirect immunofluorescence assay kit, Mosaic Basic Profile 3 (Euroimmun, Lübeck, Germany). The immunofluorescence assay was performed with an initial dilution of 1:100. In addition to serological testing, hepatitis E polymerase chain reaction was performed in 50% of the cases. Whenever a liver biopsy was conducted during the diagnostic work-up, the results were extracted from the pathological report.

Statistical analyses were performed using SPSS, Version 26.0.0.1 (IBM, Armonk, NY, USA). After testing for normal distribution, parametric or non-parametric tests (Chi-square test, Fisher’s exact test or Mann–Whitney U test) were applied, p ≤ 0.05 was considered statistically significant.

From our cohort of 383 patients with acute liver injury who were included in our prospective study on potential DILI drugs owing to the initial suspicion of DILI, 238 patients were diagnosed with DILI. Out of those 238 patients, we identified 32 patients with suspected metamizole-induced DILI (13%). All of these patients had a concomitant medication: non-steroidal antirheumatic drugs were used in ten (31%) cases, antibiotics also in ten (31%) cases, anaesthetics in three (9%) cases, herbals in two (6%) cases and others in seven (22%) cases. For an overview of the concomitant medication, see Table 1 of the Electronic Supplementary Material (ESM). In all of the patients, RUCAM for metamizole was ≥ 3, meaning that metamizole was possibly, probably or highly probably the causative agent for the DILI episode. Adjudication by the treating physician revealed metamizole-induced DILI as the most likely diagnosis in eight of the patients with DILI (25%), while in the remaining 24 patients (75%) an association with metamizole as the causative agent was assessed as the most likely diagnosis but could not be proven because of the intake of concomitant medication with a compatible time to onset. The clinical characteristics of the patients with suspected metamizole-induced DILI are summarised in Table 1. Sixty-nine percent of the patients were female and the median age at diagnosis was 41 years. In the majority of cases, the patients presented with a hepatocellular type of injury (n = 26, 81%). The median RUCAM score both for metamizole and the concomitant medication with the highest RUCAM was 5 (range 3–9). Anti-nuclear antibodies were found in 72% (n = 23) of the patients, while 34% (n = 11) presented with positive antimitochondrial antibodies (AMA). Median immunoglobulin G levels were within the range of normal (13.1 g/L [5.3–24.3 g/L]; ULN: 16 g/L), none of the patients were positive for other liver-specific autoantibodies, such as anti-smooth muscle antibodies, liver-kidney microsomal antibodies, or soluble liver antigen, and none of the patients had a persistent liver injury or relapse, ruling out autoimmune hepatitis or chronic autoimmune biliary disease. The median follow-up was 5 months, which ensured that relapse of liver injury did not occur after the acute episode of DILI.

A liver biopsy was performed in 20 of the 32 patients (63%). The histopathological characteristics found in the liver specimen analysis are provided in Table 2 of the ESM. Inflammatory infiltrates were reported for every patient with a predominant infiltration with eosinophilic cells (n = 17, 85%). Further, in the majority of cases necrosis (n = 17, 85%) of mostly moderate to pronounced intensity (n = 12/17, 71%) was observed. Infiltration with plasma cells was found in 12 patients (60%). In four cases, a low-grade fibrosis was detected, while none of the patients presented with advanced fibrosis. Representative pictures of eosinophilic cell infiltration and necrosis in a liver biopsy from a patient with DILI from our cohort are shown in Fig. 1a–d. Seven patients had a positive re-challenge, meaning that they had a second episode of acute liver injury after unintentional re-exposure to metamizole alone (n = 3, 9%) or a combination of metamizole and the respective comedications (n = 4, 13%).

If patients with RUCAM ≥ 6 for metamizole (metamizole-induced DILI probable or highly probable; n = 12) and RUCAM 3–5 (metamizole-induced DILI possible; n = 20) were compared, no significant differences regarding clinical, laboratory and histopathological results were observed (Table 2). Age, sex distribution, the predominantly hepatocellular type of injury, the level of liver function test abnormalities both at the onset of disease and at peak value, severity and outcome were similar between both groups. The only difference was that patients with RUCAM < 6 presented with higher rates of AMA positivity (45% vs 17%, p = 0.024). Histopathological analysis revealed that cholestasis was observed less often in patients with RUCAM ≥ 6 for metamizole (10% vs 50%); however, this difference was not statistically significant (p = 0.051). Other histological patterns with moderately strong inflammatory infiltrates and moderate to pronounced necrosis were comparable between patients with RUCAM ≥ 6 and RUCAM < 6 (Table 3 of the ESM). A detailed overview of the characteristics of the individual patients with suspected metamizole-induced DILI cases and high RUCAM scores for metamizole is provided in Table 4 of the ESM.

Reliability of the RUCAM for a causality assessment is limited [11], especially in polymedicated patients. This is also illustrated in the current cohort: eight patients (25%) had a RUCAM of 6 or higher for both metamizole and at least one of the concomitant medications, while another six patients (19%) had RUCAM scores of 6 or higher for at least one of the concomitant drugs but not metamizole. In addition to expert opinion and RUCAM scoring as a gold standard for establishing the diagnosis, the causality assessment was supported by the MH cell test, an in vitro test system established in our centre and validated in patients with re-exposure previously [17]. The MH cell test was performed for every patient in this cohort and metamizole was tested for in 30/32 (94%) of the cases. The MH cell test showed toxicity against metamizole in 24 of those 30 patients (75%). An overview of the individual test results for metamizole is shown in Fig. 1 of the ESM. Patients with positive MH cell test results for metamizole were more likely to present with a hepatocellular type of injury (88% vs 50%, p = 0.048). Otherwise, no significant differences were observed regarding clinical or laboratory characteristics (Table 3). Histopathological patterns were also similar in patients who tested negative and positive for metamizole in the MH cell test (Table 3 of the ESM).

A special focus was placed on the 14 patients who presented with a RUCAM for the concomitant medication of at least 6. As stated above, eight of those patients also had a RUCAM for metamizole of 6 or higher. An overview over the individual patients’ MH cell test results for metamizole and the concomitant drugs is given in Table 5 of the ESM. The MH cell test results showed toxicity of metamizole but not of the comedication in seven (50%) cases and of a combination of metamizole and the comedication in three (21%) cases. In four cases (29%), the MH cell test was negative for both metamizole and the co-administered drug.

In addition to testing patients with suspected metamizole-DILI, MH cell testing has also been performed on a variety of control subjects: (a) healthy controls (n = 8), (b) patients with metamizole intake and acute liver injury caused by conditions other than DILI (n = 18), (c) patients with DILI without metamizole intake (n = 17) and (d) patients with metamizole treatment but DILI due to another drug (n = 39). Regarding the healthy controls, half had been exposed to metamizole (n = 4). The test was negative in seven of all eight healthy controls (88%) and three of the four (75%) healthy controls with prior metamizole intake. In 15 out of the 18 patients with intake of metamizole but liver injury not associated with DILI, the test showed negative results (83%). Regarding DILI patients without a prior intake of metamizole, MH cell testing yielded negative results in 34/39 cases (87%). In patients with DILI with prior metamizole therapy but in whom liver injury was highly likely associated with another drug (n = 17), the MH cell test gave negative results in 16 cases (94%). Positive results for the concomitant medication were observed in 16 of those 17 patients. One case had a positive result for both metamizole and the concomitant herbal drug and in one case the MH cell test was negative for both metamizole and the concomitant medication.

To focus on the more distinctive features of severe cases among all of the 32 cases of suspected metamizole-induced DILI, a subgroup analysis was performed comparing patients with DILI who developed a clinically relevant jaundice, i.e., peak TBIL >3 mg/dL, with patients without clinically relevant jaundice (Table 4). Regarding the basic clinical parameters, jaundiced patients with DILI-developed liver injury with a longer latency from the start of metamizole intake (58 vs 31 days, p = 0.039) presented with higher peak levels of ALT, AST, TBIL and INR, as well as higher MELD scores both at the onset of liver injury and at peak levels. Severity according to the 2011 criteria [12] was significantly higher in jaundiced patients (severe [3] vs moderate [1], p = 0.002). Moreover, coagulopathy as well as Hy’s law positivity was observed more often in jaundiced patients. This also translated into a higher occurrence of ALF, which was observed in 33% of the jaundiced patients but for none of the non-jaundiced patients (p = 0.03). Two of the jaundiced patients required high-urgency liver transplantation (10% vs 0%, p = 0.23). A liver biopsy was performed in the majority of patients with jaundice (n = 15, 71%), and in 46% of the patients without jaundice (n = 5; Table 6 of the ESM). Interestingly, the proportion of patients presenting with steatosis was significantly higher in non-jaundiced patients (60% vs 7%, p = 0.034) while cholestasis (mainly hepatocellular and canalicular) and fibrosis were only observed in liver specimens of jaundiced patients (40% vs 0%, p = 0.09; and 27% vs 0%, p = 0.20, respectively).

Seven of the 32 patients (22%) with suspected metamizole-induced DILI presented with ALF. Concomitant medications used by these patients were ibuprofen (n = 4), cefuroxime (n = 4), sevoflurane (n = 3), propofol (n = 2), diclofenac (n = 2), acetylsalicylic acid, amoxicillin/clavulanic acid, ampicillin/sulbactam, ciprofloxacin, dienogest/ethinylestradiol, pantoprazole, paracetamol, valsartan and zopiclone (each n = 1). Patients who developed ALF had a longer latency between the initiation of metamizole and the onset of liver injury (80 vs 34 days, p = 0.033) and higher levels of AST, TBIL, INR and MELD both at the onset of liver injury and at the respective peak levels as well as higher ALT levels at onset (Table 5). Patients with ALF also had a higher severity according to the 2011 criteria [12] and were more likely to present with jaundice or coagulopathy, while there was no significant difference in Hy’s law positivity (Table 5). In accordance with the higher severity of those cases, the proportion of patients needing high-urgency liver transplantation was also significantly higher (29% vs 0%, p = 0.02). Regarding histopathological analysis, patients with ALF more frequently presented with mild fibrosis (60% vs 7%, p = 0.01), while the patients who did not develop ALF showed a higher proportion of eosinophilic cell infiltration (93% vs 60%, p = 0.033; Table 6 of the ESM).